The increasing demand for new therapeutic proteins, technical enzymes, protein engineering, and functional genomics requires a rapid and efficient protein production and screening platform. Leader et al. (2008) Nat. Rev. Drug Discov. 7(1):21-39; Swartz (2012) Aiche J. 58(1):5-13. The emerging technology of cell-free protein synthesis (CFPS) can help to satisfy this demand. Carlson et al. (2012) Biotechnol. Adv. 30(5):1185-94. Compared to cell-based expression, CFPS offers advantages such as shorter process times and the direct control and monitoring of reaction conditions. Swartz (2012), supra. PCR products can be used directly for the simultaneous expression of multiple proteins without laborious cloning and transformation steps. Wu et al. (2007) Angew. Chem. Int. Ed. Engl. 46(18):3356-8; Yabuki et al. (2007) J. Struct. Funct. Genomics 8(4):173-91; Gan & Jewett (2014) Biotechnol. J. 9(5):641-51. CFPS platforms allow the addition of accessory factors that promote protein folding (Ozawa et al. (2005) J. Biomol. NMR 32(3):235-41; Endo et al. (2006) Mol. Biotechnol. 33(3):199-209; Matsuda et al. (2006) J. Struct. Funct. Genomics 7(2):93-100), or the incorporation of unnatural amino acids (Albayrak & Swartz (2013) Nucleic Acids Res. 41(11):5949-63); White et al. (2013) Methods 60:70-4). They also facilitate the expression of cytotoxic proteins that cannot be produced in living cells. Xu et al. (2005) Appl. Biochem. Biotechnol. 127(1):53-62; Schwarz et al. (2008) Proteomics 8(19):3933-46; Xun et al. (2009) Protein Expr. Purif. 68(1):22-7.
Escherichia coli cell-free lysates are widely used and are advantageous because of their low cost, scalability, and high productivity. Zawada et al. (2011) Biotechnol. Bioeng. 108(7):1570-8; Caschera & Noireaux (2014) Biochimie 99:162-8. However, because the lysates originate from bacteria, they are unsuitable for the production of complex proteins with multiple subdomains due to inefficient oxidative folding, and the absence of chaperones and glycosylation machinery. Eukaryotic cell-free systems are better suited for the expression of such proteins, and support most forms of post-translational modification. Chang et al. (2005) J. Mol. Biol. 353(2):397-409; Zhang & Kaufman (2006) Handb. Exp. Pharmacol. (172):69-91. The most frequently used systems are based on wheat germ extract (WGE), insect cell extract (ICE), and rabbit reticulocyte lysate (RLL). However, these systems are expensive, and extract preparation is complex. Carlson et al. (2012), supra. This has created a demand for additional eukaryotic CFPS, such as those based on Leishmania tarentolae (Mureev et al. (2009) Nat. Biotechnol. 27(8):747-52), Chinese hamster ovary (CHO) cells (Brodel et al. (2014) Biotechnol. Bioeng. 111(1):25-36), and Saccharomyces cerevisiae (Hodgman & Jewett (2013) Biotechnol. Bioeng. 110(10):2643-54; Gan & Jewett (2014), supra).
The use of cell-free systems to perform in vitro protein synthesis has been limited, for example, by the short reaction times and low protein production rates that are characteristic of such systems. These qualities lead to poor protein yields and excessive costs per unit of protein produced.
Longer reaction times are obtainable through the use of a continuous translation reaction, using a continuous flow system. Spirin et al. (1988) Science 242:1162-1164. Continuous reactions are performed over tens (or even hundreds) of hours, and methods relying on continuous flow must constantly supply necessary reaction substrates to the chamber. Thus, these reactions require a substantial investment of time and resources. Furthermore, translation in a “continuous” system is directed towards producing large amounts of protein, and the system differs substantially from those used to perform static (“batch”) in vitro translation reactions. Static reactions can be run in a small reaction volume (e.g., microliters), and are not directed towards producing preparative amounts (e.g., milligrams) of proteins. Such batch reactions may be completed in one to two hours. For all of the foregoing reasons, while it increases the reaction duration and protein yield as compared to a corresponding batch system, a continuous reaction system requires more expensive reagents.